Xanthene dyes are among the most commonly used class of fluorescent dyes. Fluorescein, a dihydroxy xanthene dye, is a particularly useful biological dye for several reasons: the dianionic form of the dye is very soluble in water; its pK.sub.a is 6.3-6.7 thus being mostly deprotonated and fluorescent at physiological pH (7.0-7.6); the excitation maxima of the dye (490 nm) is well matched to a 488 nm line of an argon ion laser; it has a high extinction coefficient (8.times.10.sup.4 M.sup.-1 cm.sup.-1); and a high guantum yield (0.93). The last two properties allow the detection of minute guantities of the dye.
In addition to these properties, derivatives of fluorescein are readily synthesized and allow utilization of the dye for biological purposes (e.g., fluorescein diacetate has utility as a membrane permeable esterase substrate). One derivative, fluorescein isothiocyanate (FITC), has been used extensively in the field of flow cytometry.
Flow cytometers generally are characterized as having one or more sources of excitation (typically lasers tuned to a specific frequency), means which allow the passage of cells substantially one at a time through a measurement region and means for detection of light scatter and fluorescence from cells as they pass through the measurement region. Means to record the data for each of the cells also may be coupled to the device.
Fluorescein isothiocyanate typically is used in such systems coupled to a monoclonal antibody (MAb) which will react with certain cells in a sample, although it may be coupled to any type antibody. When cells bearing the MAb conjugated to FITC pass through the measurement region, the laser excites the dye and fluorescence is emitted and collected by one of the detection means. An article by Herzenberg et al., Sci. Amer., (1979) and U.S. Pat. No. 4,599,307 describe a typical flow cytometer and describe the use of FITC conjugated MAbs to detect the presence of certain cells. U.S. Pat. No. 4,727,020 further describes a method for the analysis of subpopulations of blood cells using FITC conjugated MAbs in combination with other tagging agents, such as a nucleic acid dye.
The usefulness of fluorescein isothiocyanate, however, is somewhat limited in that it is excited at 488 nm and emits at 520 nm. It would be useful to have the characteristics of FITC but with an excitation maxima to the red of fluorescein. The advantages of a dye with a longer wavelength excitation and emission are several: background fluorescence from biological materials (e.g., DNA) diminishes; the cost and size of the laser needed to excite the dye diminishes; and the possibility of dual laser excitation using an argon ion laser as the lower wavelength laser rises. As such, dyes having such properties would be useful in the analysis of cells in a sample.